Phase II year
2020
(last award dollars: 2022)
Phase II Amount
$2,304,786
Misfolded protein aggregates are toxic supramolecular proteinaceous conglomerates that self- replicate in the host, causing damage to surrounding tissue. Several of these protein aggregates are strongly associated with neurodegenerative diseases, such as the prion protein in prion diseases, Amyloid ? and tau in Alzheimers (AD), TDP-34 in amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD), and ?-synuclein (?S) in Parkinsons disease (PD), dementia with Lewy bodies (DLB) and Multiple Systemic Atrophy (MSA). The molecular mechanisms behind the in vivo formation of these aggregates and their effect on neural tissue remain elusive, but their compelling implication as early drivers of the disease processes identifies them as potentially useful disease biomarkers and therapeutic targets. The primary goal of this proposal is to perform robust analytical validation of a Protein Misfolding Cyclic Amplification (PMCA) assay for detection of ?S aggregates in cerebrospinal fluid (CSF) and plasma of patients with synucleinopathies. The PMCA platform uses the intrinsic self-replicating nature of misfolded protein aggregates to cyclically amplify minute quantities of ?S aggregates in biological samples. The assay is performed in a 96-well plate, and the amplification is monitored over time using the fluorescent amyloid-specific dye, ThioflavinT. Method development is focused on robustness to support future use in a controlled laboratory with high sample throughput and strict requirements for assay accuracy and reproducibility. Analytical validation will be performed in accordance with FDA and Clinical Laboratory Standard Institute (CLSI) recommendations, using a combination of clinically positive patient samples and samples spiked with synthetically generated aggregates to evaluate method accuracy, precision, sensitivity, specificity, reportable range, and other critical assay characteristics. The overall objectives for phase I are to finalize standard operating procedures and protocols and perform analytical validation of qualitative/semi-quantitative ?S-PMCA assays for detection of ?S oligomers in CSF and plasma for use as a diagnostic biomarker for PD and related synucleinopathies. The overall objectives for phase II are to adapt the assays for quantitation/kinetic characterization of ?S oligomers in CSF and plasma, and to explore expanded context of use to include indications as: a susceptibility/risk biomarker to identify patients at risk of developing synucleinopathies prior to onset of clinical symptoms; a diagnostic biomarker to distinguish between different type of synucleinopathies; a monitoring biomarker to assess disease progression or treatment effectiveness; and a prognostic biomarker with ability to characterize disease type and severity.
Public Health Relevance Statement: Narrative Misfolded protein aggregates are strongly associated with many human neurodegenerative diseases, including ?-synuclein (?S) aggregates in Parkinson's disease (PD) and other synucleinopathies. The primary goal of this proposal is to perform robust analytical validation of a Protein Misfolding Cyclic Amplification (PMCA) assay for detection of ?S aggregates in cerebrospinal fluid (CSF) and plasma. Analytical validation will be performed in accordance with FDA and Clinical Laboratory Standard Institute (CLSI) recommendations, using a combination of confirmed positive patient samples and samples spiked with synthetically generated aggregates to evaluate method accuracy, precision, sensitivity, specificity, reportable range, and other critical assay characteristics, and generate data required for regulatory approval.
Project Terms: alpha synuclein; Alzheimer's Disease; Amyloid; Amyloid beta-Protein; Atrophic; base; Biochemical; Biological; Biological Assay; Biological Markers; Body Fluids; Calibration; Cerebrospinal Fluid; Cerebrospinal Fluid Proteins; Characteristics; Clinical; Data; Data Analyses; design; Detection; Development; Diagnosis; diagnostic biomarker; Diagnostic tests; Disease; Disease Progression; Dyes; Early Diagnosis; effectiveness evaluation; Ensure; Evaluation; frontotemporal lobar dementia-amyotrophic lateral sclerosis; Future; Goals; Human; In Vitro; in vivo; Institutes; Kinetics; Laboratories; Lewy Body Dementia; Liquid substance; method development; Methods; misfolded protein; Molecular; Monitor; Multiple System Atrophy; Nature; Neurodegenerative Disorders; Parkinson Disease; Patients; Periodicity; Phase; Plasma; Plasma Proteins; pre-clinical; Predisposition; Preparation; Prion Diseases; Procedures; Process; Prognostic Marker; protein aggregation; protein misfolding cyclic amplification; Protocols documentation; PrP; Reaction; Reagent; Recommendation; Recovery; relating to nervous system; Reproducibility; Research; research clinical testing; Research Personnel; Risk; Sampling; Sensitivity and Specificity; Severities; Specificity; Symptoms; synucleinopathy; tau Proteins; Techniques; Testing; Therapeutic Intervention; therapeutic target; time use; Tissues; Treatment Effectiveness; trend analysis; United States; Validation
